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  • br Materials and methods br Results br Discussion


    Materials and methods
    Discussion In this study, the N&B analysis showed new evidence regarding the proportion and the oligomerization state of the AT1 and the ETA receptors near or on the plasma membrane. Althought, preliminary studies suggest that the AT1 and the ETA receptors occurred only as monomers and dimers [39,40], this study showed that for both receptors the majority of the particles appeared as monomers, a small amount as dimers and a much smaller proportion as tetramers. Preliminary studies suggest that dimerization of the AT1 receptor occurred during the biosynthesis in the endoplasmic reticulum before surface expression [27,40], which may explain the presenece of these Glyoxalase I inhibitor before the addition of any ligand. Our results showed that the presence of Ang II did not change the proportion of AT1 receptor monomers, dimers, and tetramers, which is in agreement with other studies using FRET, BRET and co-immunoprecipitation techniques [27,41]. Regarding ETA receptors, preliminary studies using FRET showed that both ET-1 and BQ123 reduced ETA receptor dimers [40,42], while the present study showed that BQ123 reduced receptor-dimers and ET-1 has no influence over receptor populations. Little is still known about the effect of antagonists on the population of monomeric, dimeric and higher order aggregates of these two receptors. For instance, the AT1 receptor antagonist telmisartan did not modify the population of AT1 dimers [27], while the ETA receptor antagonist BQ123 reduced the proportion of ETA dimers [42]. Our results showed that the effect of BQ123 is similar to that found in literature, however the effect shown by the AT1 receptor antagonist losartan is different to that of telmisartan. Additionally, the ETA receptor antagonist sitaxentan had no effect on any of the ETA receptor populations. The question arises whether losartan and BQ123 have a different pharmacological effect which is responsible for the differences seen at the level of receptor organization. In this line, there is evidence that suggests that both, losartan and BQ123 act as inverse agonists [43]. Regarding to the variations in the monomer/dimer/tetramer rate among cells of CHO-ETAEGFP receptors before adding the ligands, we hypothesize the used of transient transfection increase the variability further experiments need to be done to clarify this observations. In several studies the ability of the ligands to modify receptor oligomerization have been investigated. For instance, agonists promoted association of the receptors, in others the formation of oligomers is constitutive, or they dissociate upon treatment with the agonist, antagonist and inverse agonist. [[44], [45], [46], [47], [48], [49], [50]]. Caution must be taken when selecting the experimental approach to monitor the organization state of membrane receptors. For example, available biochemical and biophysical techniques [51] usually present pitfalls such as: artificial protein–protein associations due to the lysis and solubilization of cell material, impossibility to distinguish between interactions at the cell surface and interactions that occur in intracellular compartments, low signal-to-noise ratio associated with FRET imaging and the need of specific dyes to overlap between the donor emission spectrum and the acceptor absorption spectrum. [27,42,52,53]. Therefore, this study proposes the N&B analysis coupled to TIRFM as a suitable method to discriminate monomeric from higher order oligomeric receptors located on and near the plasma membrane without any influence from the receptor concentration. Even though the molecules are slowly diffusing or interacting with different cellular features as arrestin, lipid raft domains or are in the processes of desensitization, internalization of the receptor-ligand complexs, the use of fast cameras with high frame rates allow to detect each species [33,54]. Additionally, this study supports the use of a detrending algorithm to avoid the overestimation of the brightness produced by photobleaching and cell movement, obtaining a more realistic images of the aggregates [30].